Pathogenic DICER1 RNase IIIb Hotspot Mutation Induces 3p-miRNA Gain-of-Function via Argonaute Strand Switch [miR-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE279556 | GEO | 2025/07/18
REPOSITORIES: GEO
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Project description:Dicer is an essential enzyme in microRNA biogenesis. Mutations in the DICER1 gene are linked to various cancers, notably through the DICER1 syndrome. To investigate the impact of the pathogenic hotspot mutations in DICER1-associated tumors, we introduced a hotspot mutation into the endogenous Dicer1 locus of a mouse embryonic carcinoma cell line using CRISPR. Our findings not only confirm the loss of 5p-miRNAs, as previously reported, but also uncover an unexpected upregulation of specific 3p-miRNAs. These upregulated 3p-miRNAs, usually considered as passenger strands in the wild-type cells, are selectively loaded into the Argonaute protein in mutant cells based on their 5' end characteristics, resulting in a "strand-switch" phenomenon. Functional assays and transcriptome analyses demonstrate the passenger 3p-miRNAs’ activity. This study suggests that the Dicer hotspot mutation is not merely a loss-of-function mutation for 5p-miRNAs but also a gain-of-function mutation for passenger 3p-miRNA, potentially contributing to DICER1-associated tumorigenesis.
2025-07-18 | GSE279636 | GEO
Project description:Dicer is an essential enzyme in microRNA biogenesis. Mutations in the DICER1 gene are linked to various cancers, notably through the DICER1 syndrome. To investigate the impact of the pathogenic hotspot mutations in DICER1-associated tumors, we introduced a hotspot mutation into the endogenous Dicer1 locus of a mouse embryonic carcinoma cell line using CRISPR. Our findings not only confirm the loss of 5p-miRNAs, as previously reported, but also uncover an unexpected upregulation of specific 3p-miRNAs. These upregulated 3p-miRNAs, usually considered as passenger strands in the wild-type cells, are selectively loaded into the Argonaute protein in mutant cells based on their 5' end characteristics, resulting in a "strand-switch" phenomenon. Functional assays and transcriptome analyses demonstrate the passenger 3p-miRNAs’ activity. This study suggests that the Dicer hotspot mutation is not merely a loss-of-function mutation for 5p-miRNAs but also a gain-of-function mutation for passenger 3p-miRNA, potentially contributing to DICER1-associated tumorigenesis.
2025-07-18 | GSE279645 | GEO
Project description:Recurrent somatic hotspot mutations of DICER1 appear to be clustered around each of four critical metal binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3’ end of the 5p-miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated “hotspot” mutations we engineered mouse Dicer1-deficient ES cells to express wild-type and an allelic series of the mutant human DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal binding site mutations were compared to each other and wild-type human DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation carrying cells were distinct from both wild-type and Dicer1-deficient cells. Further, miRNA profiles showed mutant DICER1 results in a dramatic loss in processing of mature 5p-miRNA strands but were still able to create 3p-strand miRNAs. Messenger-RNA profile changes were consistent with the loss of 5p-strand miRNAs and showed enriched expression for predicted targets of the lost 5p derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA-processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p-strand miRNA. We further propose that this resulting 3p-strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. A total of 28 Affymetrix Mouse Gene ST arrays were done for mRNA expression profiling of various DICER1 mutants (n=14), wildtype controls (n=6), vector only (n=3) and parental cell lines (n=5).
2012-10-01 | E-GEOD-40965 | biostudies-arrayexpress
Project description:Recurrent somatic hotspot mutations of DICER1 appear to be clustered around each of four critical metal binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3’ end of the 5p-miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated “hotspot” mutations we engineered mouse Dicer1-deficient ES cells to express wild-type and an allelic series of the mutant human DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal binding site mutations were compared to each other and wild-type human DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation carrying cells were distinct from both wild-type and Dicer1-deficient cells. Further, miRNA profiles showed mutant DICER1 results in a dramatic loss in processing of mature 5p-miRNA strands but were still able to create 3p-strand miRNAs. Messenger-RNA profile changes were consistent with the loss of 5p-strand miRNAs and showed enriched expression for predicted targets of the lost 5p derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA-processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p-strand miRNA. We further propose that this resulting 3p-strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations.
2012-10-01 | GSE40965 | GEO
Project description:DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic “hotspot” mutations at four mental binding sites within the RNase IIIb domain of DICER1, were identified in ovarian sex cord-stromal tumors and have since been described in other pediatric tumors. In this study, we identified and characterized DICER1 hotspot mutations in endometrial cancers derived from The Cancer Genome Atlas (TCGA) and our local tumor bank. DICER1 hotspot mutations are found in ~2% of endometrial tumors. Using Illumina and Sanger targeted resequencing we observed biallelic DICER1 mutations in more than 50% of cases with hotspot mutations and identified an additional recurrent mutation G1809R in 2 cases. Through small RNA deep sequencing and real-time PCR, we demonstrated mutations that add a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production as mutations at metal binding sites. In one case G1809R was compound heterozygous with a germline S839F mutation, which contributes to loss of DICER1 expression by promoting protein degradation. As expected, 5p miRNAs are globally reduced in tumors and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes derepressed due to loss of 5p miRNAs are strongly associated with cell cycle related pathways. Using a Dicer null cell line model, we demonstrated that DICER1 hotspot mutants abolished the inhibitory effects of wildtype DICER1 on cell proliferation upon re-expression. Furthermore, targets of let-7 family miRNAs are enriched among the upregulated genes, suggesting loss of let-7 may be impacting downstream pathways.
2015-03-16 | GSE65092 | GEO
Project description:Purpose: Identify whether recombinant ncRNAs are recognized by mammalian cells and processed into mature microRNAs, thus leading to the alteration of downstream target genes. In addition, we also explored whether cleavage is Dicer dependent. Methods: HEK293T (Dicer-WT) and 424 (Dicer-KO) cell lines were treated with 20nM of control tRNA (MSA), nCAR/miR-34a-5p, or nCAR/miR-34a-3p in triplicate. Results: Read counts of known miRNAs were analyzed using a miRDeep module, and read counts derived from nCAR/miR-34a-5p or nCAR/miR-124a-3p transfection were obtained by mapping sequence reads by an in-house developed PERL script. EdgeR was used to analyze significant (P-value < 0.05 and log2CPM > 6) differentially expressed miRNAs and sRNAs. Conclusions: Upon transfection of recombinant RNA agents in mammalian cells, nCAR/miR-34a-5p became the most dominant miRNA and was processed in a Dicer dependent manner, while nCAR/miR-124a-3p become the 7th most abundant miRNA and displayed comparable levels with or without Dicer. In both cases, different isoforms of the mature miRNA sequence were generated.
2020-09-01 | GSE103349 | GEO
Project description:MiRNAs have been shown to alter both protein expression and secretion in different cellular contexts. By combining in vitro, in vivo and in silico techniques, we demonstrated that overexpression of pre-miR-1307 reduced the ability of breast cancer cells to induce endothelial cell sprouting and angiogenesis. However, the molecular mechanism behind this and the effect of the individual mature miRNAs derived from pre-miR-1307 on protein secretion and is largely unknown. Here, we overexpressed miR-1307-3p|0, -3p|1 and 5p|0 in MDA-MB-231 breast cancer cells and assessed the impact of miRNA overexpression on protein secretion by Mass Spectrometry. Unsupervised hierarchical clustering revealed a distinct phenotype induced by overexpression of miR-1307-5p|0 compared to the controls and to the 5’isomiRs derived from the 3p-arm. Together, our results suggest different impacts of miR-1307-3p and miR-1307-5p on protein secretion which is in line with our in vitro observation that miR-1307-5p, but not the isomiRs derived from the 3p-arm reduce endothelial cell sprouting in vitro. Hence these data support the hypothesis that miR-1307-5p is at least partly responsible for impaired vasculature in tumors overexpressing pre-miR-1307.
2024-12-11 | PXD041087 | Pride
Project description:WNT signaling is fundamental to bone health and its aberrant activation leads to skeletal pathologies. A heterozygous missense mutation p.C218G in WNT1, a key WNT pathway ligand, leads to severe early-onset osteoporosis with multiple peripheral and spinal fractures. Despite the severe skeletal manifestations, conventional bone markers are normal in mutation-positive patients. The objective of this study was to find novel biomarkers that differentiate between WNT1 mutation-positive and -negative subjects. We evaluated serum levels of 192 miRNAs in 12 mutation-positive (median age 39 years, range 11-76 years) and 12 mutation-negative (35 years, range 9-59 years) subjects from two Finnish families. The results indicate significant differences in circulating miRNA profiles with 2 upregulated (miR-18a-3p, miR-223-3p) and 6 downregulated miRNAs (miR-22-3p, miR-31-5p, miR-34a-5p, miR-143-5p miR-423-5p, miR 423-3p) in the mutation-positive subjects. Three of these (miR-22-3p, miR-34a-5p, and miR-31-5p) are known inhibitors of WNT signaling: miR-22-3p and miR-34a-5p target WNT1 mRNA and miR-31-5p is predicted to bind to the WNT1 3’UTR. Our results suggest that the WNT1 mutation disrupts a feed-back regulation between these miRNAs and WNT1, providing new insights into the pathogenesis of WNT-related bone disorders. Future studies are warranted to explore the potential diagnostic and therapeutic applications of these findings in osteoporosis.
2018-03-29 | GSE103473 | GEO
Project description:miR-493-5p, miR-3662, and miR-589-3p were estimated as working microRNAs in bleomycin- and methotrexate-induced phenotypic changes in A549 cells via microRNAs-Proteins Analysis of Integrative Relationship (miR-PAIR). To verify the effect of these miRNAs on the their target protein expression levels, comprehensive expression of proteins in A549 cells treated with miR-493-59, miR-3662, and miR-589-3p mimic was examined by SWATH-MS method. As expected by a miR-PAIR mehtod, almost target proteins were succesfully regulated by miR-493-5p and miR-589-3p mimics.
2025-07-25 | PXD053397 | Pride
Project description:MiRNAs regulate posttranscriptional gene expression and are widely implicated in the pathogenesis of complex diseases. We aim to elucidate miRNA regulation of the atrial mRNA signatures that associate with AF. This may provide novel mechanistical insights and candidate targets for therapies using miRNA mimics or antimiRs.
We present combined miRNAs-mRNAs sequencing in atrial tissues of patient without AF (n=22), with paroxysmal AF (n=22) and with persistent AF (n=20). MiRNA and mRNA signatures followed an ordinal scale from nonAF to paroxysmal to persistent AF patients. The previously reported mRNA sequencing identified 5228 differentially expressed genes involved in epithelial to mesenchymal transition, endothelial cell proliferation and extracellular matrix remodelling involving collagens, glycoproteins and proteoglycans. We discovered 103 differentially expressed miRNAs. Key downregulated miRNAs included miR-135b-5p, miR-138-5p, miR-200a-3p, miR-200b-3p and miR-31-5p and key upregulated miRNAs were miR-144-3p, miR-15b-3p, miR-182-5p miR-18b-5p, miR-4306 and miR-206. The expression levels of differentially expressed miRNAs were negatively correlated with the expression levels of their predicted target mRNAs. The downregulated miRNAs demonstrated a more profound transcriptome effect than the upregulated miRNAs. Upregulated biological processes enriched in miRNAs targets related to epithelial and endothelial cell migration and glycosaminoglycan biosynthesis, in line with the processes discovered by the mRNA sequencing analysis.
Combined analysis of miRNA and mRNA sequencing uncovered miRNAs with a broad transcriptional effect in human AF. Epithelial to mesenchymal transition and endothelial cell proliferation were processes controlled by downregulated miR-135b-5p, miR-138-5p, miR-200a-3p, miR-200b-3p and miR-31-5p, which in turn may contribute to (myo)fibroblast activation and structural remodeling.\
| EGAS00001005296 | EGA